Abstract:

As for many genetic disorders, the novel Base Editing system derived from the CRISPR/Cas9 system, and its ability to mediate genome editing serves a potential tool for correcting the point mutation largely responsible for type 1 Gaucher disease, N370S. We conducted the present study to determine whether Base Editor-mediated targeting at N370S within the GBA1 gene was feasible in human induced pluripotent stem cells (iPSCs). Cytidine base editor and guide RNA (gRNA) plasmids were transformed into E.coli cells for the production of many plasmids. Plasmids were then isolated from E.coli and delivered into patient-derived human iPSCs by electroporation. Under fluorescence microscopy, successful transfection was represented by cells fluorescing green, as the cytidine base editor plasmid expressed green fluorescent protein. To screen for cells that had undergone correction, mismatch primers were designed to introduce an HpyCH4III restriction endonuclease sequence, whereby only wildtype DNA is recognized and cleaved. A proof-of-concept restriction digest revealed that only wildtype DNA was cleaved, while N370S DNA remained un-cleaved. RNA-guided engineered nuclease (RGEN)-restriction fragment length polymorphism (RFLP) analysis was performed to test the targeting efficiency of our gRNA to the N370S locus. The RGEN-RFLP showed cleavage by Cas9 only in the N370S condition, indicating successful targeting by the gRNA. More experimentation is necessary to determine whether cells that have acquired plasmids, as represented by green fluorescence, are in fact undergoing correction, using the HpyCH4III restriction digest, and Sanger sequencing.